The present invention relates to methods of screening for molecules, including drugs, that target and inhibit specific proteins or cellular pathways that affect the proliferation, growth or survival of a cell or organism. The methods are based on a co-culture assay, and can be applied to bacteria, yeast, C. elegans, and cultured cells, such as mammalian, insect and plant cells.
Co-culture experiments have been utilized extensively to identify genes that contribute to the fitness of cells. Giaever et al. (1999, Nat. Genet. 21:278-283) recently showed that from a large pool of cells with distinct genotypes, cells could be identified that had slight differences in fitness when grown in the presence of inhibitors. The genotypes that were responsible for the altered fitness were heterozygous mutations in diploid cells. Thus, this technique was sensitive enough to identify changes in fitness that resulted from the difference between one and two copies of a given gene.
It has been shown that overexpression of a drug""s target protein in a cell confers resistance to the cell against the drug. The resistance conferred by overexpression of a target gene has been used as a basis for screening yeast populations transformed with an expression plasmid library for yeast colonies that are resistant to tunicamycin, compactin or ethionine (Rine et al., 1983, Proc. Natl. Acad. Sci. USA 80:6750-6754; Launhardt et al., 1998, Yeast 14:935-942). The ability of a colony to grow after treatment with a drug indicates that the plasmid harbored by the colony directs expression of a protein that confers resistance to the drug, i.e., that the protein is the target of that drug.
Identification of targets for drug development is a laborious process that has had a low rate of success. Accordingly, there is a need in the art for novel methods for the development of novel drugs and therapies that modulate specific cellular pathways. The present invention provides a method for screening for compounds which specifically inhibit such target pathways. Traditional methods for identifying inhibitors of specific cellular targets typically involve in vitro assays that can directly measure the biochemical activity of an enzyme or the binding of a ligand to a receptor. Alternative methods for identifying inhibitors utilize reporter genes in intact cells that are up- or down-regulated when a specific process has been modulated in the cell by a test compound. While these approaches have been successfully used to identify pharmaceutical lead compounds, they require a considerable amount of lead time and labor to develop prior to screening thousands to hundreds of thousands of chemical compounds or natural products.
Similarly, new antibiotics are desperately needed. The widespread use of antibiotics over the past half century has lead to the emergence of bacterial strains that are resistant to nearly all antibiotics now in use. Thus there is an immediate need to develop fast and efficient methods for producing new antibiotics to combat the increasing number of these antibiotic-resistant strains (Chopra et al., 1997, Antimicrob. Agents Chemother., 37:1563-1571; Cohen, 1992, Science, 257:1050-1055; Kunin, 1993, Ann. Intern. Med., 118:557-561; Neu, 1992, Science, 257:1064-1073; Tenover and Hughes, 1996, JAMA, 275:300-304).
Traditional approaches to antibiotic development have failed to meet these needs. One commonly used approach involves chemical modification of an existing antibiotic to produce a more potent formulation. Another approach involves screening for compounds that target the resistance mechanism of a known antibiotic. Such compounds are then be used in conjunction with the known antibiotic to improve its efficacy. These approaches have been somewhat successful, but are research intensive and such drugs tend to target the same bacterial processes as existing antibiotics, and thus, like the earlier breed of antibiotics, are likely to quickly encounter resistance. A second approach has involved mass screening of compounds for their ability to inhibit bacterial growth. Using microbiological assays, natural products and semisynthetic or synthetic chemicals are screened for their ability to kill or arrest the growth of a target pathogen. At least initially, this approach has the advantage of being simple and relatively inexpensive, and allowing rapid testing of large libraries of compounds. However, the promising lead compounds that emerge from such screens subsequently must be tested for host toxicity. Furthermore, since such screens are result-oriented and blind to mechanism, further studies must be done in order to precisely understand the drug""s mechanism of action and to identify its target in the cell.
The genomes of several pathogenic microorganisms, such as Escherichia coli, Helicobacter pylori, and Chlamydia trachomatis, recently have been sequenced (Blattner et al., 1997, Science 277: 1453; Tomb et al., 1997, Nature, 388: 539-547). The availability of gene sequences encoding all proteins of these bacteria provides an unprecedented opportunity for understanding and manipulating bacterial genomes at the molecular level. A number of genes are known or are suspected to be essential to growth, survival or virulence. Such genes could be ideal targets in screening for novel antibiotics.
The present invention provides screening methods for the identification of drugs or antibiotics that target specific proteins using co-culture methods.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
The present invention provides methods for screening for a molecule that inhibits the expression or activity of a protein encoded by a target gene which affects the fitness of a cell. The methods comprise co-culturing a first cell and a second cell, wherein the first cell has higher expression or activity of the protein encoded by the target gene (xe2x80x9ctarget proteinxe2x80x9d) than the second cell, and wherein the first cell further comprises and expresses a reporter gene that is substantially not expressed in said second cell and wherein the first cell and second cell are of the same species and cell type, wherein said target protein affects the fitness of the first cell and second cell, wherein the first cell further comprises and expresses a reporter gene that is substantially not expressed in said second cell, and wherein the first cell and second cell are of the same species and cell type; and measuring the activity or amount of protein encoded by the reporter gene, wherein the activity or amount of protein encoded by the reporter gene is indicative of whether the test molecule inhibits the target gene.
In certain specific embodiments, the first and second cells are selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, a mammalian cell and a plant cell.
In an alternative embodiment, the first and second cells can be groups of cells, e.g., individual multicellular organisms of the same species. In a preferred mode of the embodiment, the species is C. elegans. 
In one embodiment, the first cell has wild-type levels of target protein expression or activity and the second cell has reduced levels of target protein expression or activity relative to wild type levels of expression or activity. In an alternative embodiment, the first cell has elevated levels of target protein expression or activity relative to wild type levels of expression or activity and the second cell has wild-type levels of target gene expression or activity. In another alternative embodiment, the first cell has elevated levels of target protein expression or activity relative to wild type levels of expression or activity and the second cell has reduced levels of target protein expression or activity relative to wild type levels of expression or activity.
The reduced level of target protein expression or activity can be generated by one copy of the target gene in a diploid cell or mutating the target gene to reduce its activity, by expressing a dominant negative form of a component of a cellular pathway of the target gene, or by lowering the activity or abundance of a target gene encoded RNA. The activity or abundance of a target gene encoded RNA can be lowered, for example, by means of a ribozyme, an anti-sense nucleic acid, a double-stranded RNA or an aptamer.
In a specific embodiment, the elevated level of target gene expression can be generated by recombinantly expressing the target gene from a plasmid or from a chromosome. The elevated level of target gene activity can also be generated by expressing a constitutively active form of the target gene.
In certain embodiments, the reporter gene of the invention encodes an enzyme, a protein or peptide comprising an epitope, a receptor, a transporter, tRNA, rRNA, or a bioluminescent, chemiluminescent or fluorescent molecule. In a specific embodiment, the fluorescent molecule is GFP or a mutant thereof. In a preferred mode of the embodiment, the fluorescent molecule is a mutant GFP having an altered fluorescence wavelength, increased fluorescence, or both. In certain specific embodiment, the mutant GDP is blue GFP. In other modes of the embodiment, the fluorescent molecule is red fluorescent protein or yellow fluorescent protein.
In certain specific embodiments, the first and second cells are co-cultured initially (upon establishment of the co-culture) at a ratio of 1:1, 1:10, 1:100, 1:1000, or 1:10000.
In certain specific embodiments, a screen of the invention is xe2x80x9cmultiplexedxe2x80x9d, i.e. one round of screening is used to identify inhibitors of multiple target genes. In such embodiments, screening comprises co-culturing a first cell, and two or more second cells, wherein each said second cell has elevated expression or activity of a different target gene than does the first cell, wherein each target gene positively contributes to the fitness of the first and second cells, wherein said second cells each further comprises and expresses a reporter gene that is substantially not expressed in said first cell, and wherein said first cell and said second cells are of the same species and cell type; exposing the co-culture to a test molecule; and detecting whether a differential sensitivity to the molecule exists between the first cell and one or more of the second cells by detecting an increase in the ratio of cells having reporter gene activity by measuring the activity or amount of protein encoded by the reporter genes in said second cells, wherein said increase in reporter gene activity indicates that the molecule inhibits one or more of said target genes.
In certain modes of multiplexing, the second cells comprise and express the same reporter gene. To determine which of the second cells have increased reporter activity in co-cultures having a significant increase in reporter activity, a secondary round of screening, or a re-screening is carried out. The re-screening can entail polymerase chain reaction (PCR), auxotrophic growth selection, or a co-culture method according to the methods of the present invention. In other modes of multiplexing, the second cells each comprises and expresses a different reporter gene.
In certain specific embodiments, a screen of the invention is used to identify a molecule that inhibits a protein encoded by a first target gene that positively contributes to cell fitness but not the protein encoded by a second, functionally similar target gene. The functionally similar target gene can be a homolog of the target gene from another species, or encode a related protein from the same species. Such related proteins include but are not limited to isozymes, splice variants, or point mutants. Such a method comprises co-culturing a first cell and a second cell, wherein the first cell expresses elevated levels of the target protein and the second cell expresses elevated levels of the protein encoded by the functionally similar gene, wherein said target gene and functionally similar gene both positively contribute to the fitness of the first cell and second cell, wherein the first cell further comprises and expresses a reporter gene that is substantially not expressed in said second cell, and wherein the first cell and second cell are of the same species and cell type; exposing the co-culture to a test molecule; and measuring the activity or amount of protein encoded by the reporter gene, wherein said increase in reporter gene activity indicates that the molecule inhibits the target gene but not the functionally similar gene.
The screening methods of the invention identify compounds that are lead candidates for drugs that cause loss of function of a target gene. There are myriad instances where specific loss of function of a gene is therapeutically desirable, where loss of function results in a desirable phenotype, e.g., in disorders involving low cholesterol levels, cancer, etc. For example, an inhibitor specific to COX2, an oncogene, a cholesterol synthesis enzyme, etc. would be desirable.
The present invention further provides a kit comprising in one or more containers a first cell and a second cell, wherein the first cell has higher expression or activity of the target gene than the second cell, wherein the first cell further comprises and expresses a reporter gene encoding a bioluminescent, chemiluminescent or fluorescent molecule that is substantially not expressed in said second cell and wherein the first cell and second cell are of the same species and cell type.
The present invention further provides an assay system comprising a first cell and a second cell, wherein the first cell has higher expression or activity of the target gene than the second cell, wherein the first cell further comprises and expresses a reporter gene encoding a bioluminescent, chemiluminescent or fluorescent molecule that is substantially not expressed in said second cell and wherein the first cell and second cell are of the same species and cell type.